MyJove CorporationJournal of Visualized Experiments1940-087X912014926Fluorescence Imaging with One-nanometer Accuracy (FIONA)10.3791/51774ENYongWangDepartment of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-ChampaignEnCaiDepartment of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-ChampaignJanetSheungDepartment of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-ChampaignSang HakLeeDepartment of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-ChampaignKai WenTengCenter for the Physics of Living Cells, University of Illinois at Urbana-Champaign; Center for Biophysics and Computational Biology, University of Illinois at Urbana-ChampaignPaul R.SelvinDepartment of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign; selvin@illinois.eduJournal ArticleVideo-Audio Media10.3791/51774Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.